Biosurfactants are produced by a variety of microorganisms most of which are bacteria. Their ability to reduce surface and interfacial tensions makes them suitable for environmental applications where hydrophobic compounds are concerned. The biodegradability and lower toxicity of biosurfactants compared to chemical surfactants is ecologically valuable. Four biosurfactant producing bacteria (Alcanivorax borkumensis DSM 11573, Bacillus subtilis DSM 3256, Bacillus licheniformis RS1, and Rhodococcus ruber DSM 43338) were investigated in this study. Their growth and biosurfactant producing profiles were studied. The Bacillus species produced exogenous biosurfactants which were extracted and identified as surfactin and lichenysin. The other isolates produced cell bound biosurfactants and were therefore selected for augmentation in hydrocarbon degradation. Bacterial bioluminescent biosensor derived quantitative structure activity relationships were employed as a tool to validate the suitability of the extracted biosurfactants as solvents for ecotoxicity assessment of hydrophobic organic compounds. The relationships obtained in biosurfactants and water did not differ (p > 0.05) and suggests that the biosurfactants did not compromise the performance of the biosensors. Remediation of diesel hydrocarbons by degraders in the presence of Alcanivorax borkumensis or Rhodococcus ruber was tested. Degradation was 5 and 3 times more effective with respiration rates 20 and 5 times higher in the presence of A. borkumensis and R. ruber respectively. The effect of biosurfactants on bioactivity in historically contaminated soils was evaluated using the extracted lichenysin. Bioactivity was improved in the presence of the biosurfactant. Bioactivity was correlated to biodegradation of hydrocarbons in a crude oil impacted soil amended with degraders, biosurfactant producers and a chemical dispersant (p < 0.001). The lowest CFU counts for heterotrophs and degraders were observed for the chemical dispersant. The results from this study further highlight the value of biosurfactants for environmental application. Improvement in production is necessary to encourage widespread commercial application of biosurfactants.